Continuous flow chamber device for separation, concentration, and/or purification of cells

ABSTRACT

The present invention relates to methods and apparatuses for cell separation. In particular, the invention relates to separation of a particular cell type from a mixture of different cell types based on the differential rolling property of the particular cell type on a substrate coated with molecules that exhibits adhesive property with the particular cell type. This technology is adaptable for use in implantable shunts and devices for cell trafficking or tumor neutralization.

This application claims priority of U.S. Provisional Patent ApplicationNos. 60/696,797, filed Jul. 7, 2005; 60/682,843, filed May 20, 2005; and60/645,012, filed Jan. 21, 2005; which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to methods and apparatuses for cellseparation. In particular, the invention relates to separation of aparticular cell type from a mixture of different cell types based on thedifferential rolling property of the particular cell type on a substratecoated with molecules that exhibits adhesive property with theparticular cell type.

BACKGROUND OF THE INVENTION

Purified cell populations have many applications in biomedical researchand clinical therapies (Auditore-Hargreaves et al., Bioconjug. Chem.5:287-300, 1994; and Weissman, Science 287:1442-1446, 2000). Often,cells can be separated from each other through differences in size,density, or charge. However, for cells of similar physical properties,separation is often accomplished by exploiting differences in thepresentation of molecules on the cell surface. Cell-affinitychromatography is based on this approach, most often by employingimmobilized antibodies to specific cell surface antigens. Such affinitycolumn separations require several distinct steps including incubationof the cells with the antibody, elution of the cells, cell collection,and release of the conjugated antibody, with each step reducing theoverall yield of cells and increasing the cost of the process.

There exists a need for obtaining cellular samples from donors that areenriched in desired biological targets. Because a heterogeneous samplemay contain a negligible amount of a biological entity of interest, thelimits of separation methods to provide viable and potent biologicaltarget in sufficient purity and amount for research, diagnostic ortherapeutic use are often exceeded. Because of the low yield afterseparation and purification, some cell-types, such as stem cells,progenitor cells, and immune cells (particularly T-cells) must be placedin long-term culture systems under conditions that enable cell viabilityand clinical potency to be maintained and under which cells canpropagate (cell expansion). Such conditions are not always known toexist. In order to obtain a sufficient amount of a biological target, alarge amount of sample, such as peripheral blood, must be obtained froma donor at one time, or samples must be withdrawn multiple times from adonor and then subjected to one or more lengthy, expensive, and oftenlow-yield separation procedures to obtain a useful preparation of thebiological target. Taken together, these problems place significantburdens on donors, separation methods, technicians, clinicians, andpatients. These burdens significantly add to the time and costs requiredto isolate the desired cells.

Stem cells are capable of both indefinite proliferation anddifferentiation into specialized cells that serve as a continuous sourcefor new cells that comprise such tissues as blood, myocardium and liver.Hematopoietic stem cells are rare, pluripotent cells, having thecapacity to give rise to all lineages of blood cells (Kerr,Hematol./Oncol. Clin. N. Am. 12:503-519, 1998). Stem cells undergo atransformation into progenitor cells, which are the precursors ofseveral different blood cell types, including erythroblasts,myeloblasts, monocytes, and macrophages. Stem cells have a wide range ofpotential applications, particularly in the autologous treatment ofcancer patients.

Typically, stem cell products (true stem cells, progenitor cells, andCD34+ cells) are harvested from the bone marrow of a donor in aprocedure, which may be painful, and requires hospitalization andgeneral anesthesia (Recktenwald et al., Cell Separation Methods andApplications, Marcel Dekker, New York, 1998). More recently, methodshave been developed enabling stem cells and committed progenitor cellsto be obtained from donated peripheral blood or peripheral bloodcollected during a surgical procedure.

Progenitor cells, whether derived from bone marrow or peripheral blood,can be used to enhance the healing of damaged tissues (such asmyocardium damaged by myocardial infarction) as well as to enhancehematologic recovery following an immunosuppressive procedure (such aschemotherapy). Thus, improved approaches to purify stem cells ex vivo,or to “re-address” circulating stem cells in vivo, has great potentialto benefit the public health.

Hematopoietic stem and precursor cells (HSPC) are able to restore thehost immune response through bone marrow transplantation, yet the demandfor these cells far exceeds the available supply. HSPC also show greatpromise for treatment of other hematological disorders. HSPC arebelieved to adhesively roll on selectins during homing to the bonemarrow in a manner analogous to the (much better understood) process ofleukocyte trafficking. Previous work has demonstrated that CD34+ cells(showing a marker of stem cell immaturity) roll more slowly and ingreater numbers than more differentiated CD34− cells. By exploiting thisdifference in rolling affinity it should be possible to construct a flowchamber device for continuous separation and purification of CD34+ cellsfrom an initial mixture of blood cells, while maintaining viability ofthe cells for subsequent use in clinical applications. Such a processwould hold several distinct advantages over current affinity columnmethods. The feasibility of cell separation based on rolling affinityhas been demonstrated only for artificial adhesive microbeads, but notfor live stem cell populations.

CD34 is a surface marker of stem cell immaturity. Recent work has shownthat CD34+ cells from the adult bone marrow and fetal liver roll moreslowly and to a greater extent on P- and L-selectin, compared to CD34−cells (Greenberg et al., Biophys. J. 79:2391-2403, 2000). Further,Greenberg et al. (Biotechnol. Bioeng. 73:111-124, 2001) demonstratedthat rolling affinity-based separations of carbohydrate-coatedmicrospheres is possible. However, there remains a need for methods andapparatus for separation of a particular type of cells, particularly,immature stem cells from other cells, such as more mature cells, in acontinuous, single-pass, high-throughput flow chamber.

SUMMARY OF THE INVENTION

Applicants have discovered a novel method and apparatus for continuousseparation or purification of cells by taking advantage of differentialrolling velocities of different cell types. Generally, cells rolls atabout the same velocity on a surface; however, applicant have discoveredthat if a surface is rendered “sticky” to a particular cell type whilenot affecting other cells, the particular cell type exhibits a differentrolling velocity and the other cells. By taking advantage of thedifference in rolling velocity, the particular cell type can beseparated, concentrated, or purified from a cell mixture.

The advantage of the present invention is that it requires fewer stepsand subjects the cells to a more physiologically relevant environment,as opposed to the artificial and harsh environment utilized by currentother methods of cell separation. The present invention does not useexpensive purified antibodies, and is cheaper, faster, and moreefficient. The present device will enable physicians to treat cancers,immunodeficiency, hematological, and, potentially, cardiac diseases withgreater efficacy.

The device of the present invention contains a surface for cell rolling,wherein the surface has been coated with a substance that chemically orphysically adheres to the type of cell being separated, concentrated, orpurified (the desired cells). In use, a mixture of cells is allowed toflow along the surface. Because the desired cells roll at a differentvelocity than the other cells in the mixture due to the adhesion betweenthe desired cells and the coated surface, it can be separated,concentrated, or purified from the other cells.

The adhesion molecule may be specific for a region of a protein, such asa prion, a capsid protein of a virus or some other viral protein, and soon. A target specific adhesion molecule may be a protein, peptide,antibody, antibody fragment, a fusion protein, synthetic molecule, anorganic molecule (e.g., a small molecule), or the like. In general, anadhesion molecule and its biological target refer to aligand/anti-ligand pair. Accordingly, these molecules should be viewedas a complementary/anti-complementary set of molecules that demonstratespecific binding, generally of relatively high affinity. Cell surfacemoiety-ligand pairs include, but are not limited to, T-cell antigenreceptor (TCR) and anti-CD3 mono or polyclonal antibody, TCR and majorhistocompatibility complex (MHC)+antigen, TCR and super antigens (forexample, staphylococcal enterotoxin B (SEB), toxic shock syndrome toxin(TSST), etc.), B-cell antigen receptor (BCR) and anti-immunoglobulin,BCR and LPS, BCR and specific antigens (univalent or polyvalent), NKreceptor and anti-NK receptor antibodies, FAS (CD95) receptor and FASligand, FAS receptor and anti-FAS antibodies, CD54 and anti-CD54antibodies, CD2 and anti-CD2 antibodies, CD2 and LFA-3 (lymphocytefunction related antigen-3), cytokine receptors and their respectivecytokines, cytokine receptors and anti-cytokine receptor antibodies,TNF-R (tumor necrosis factor-receptor) family members and antibodiesdirected against them, TNF-R family members and their respectiveligands, adhesion/homing receptors and their ligands, adhesion/homingreceptors and antibodies against them, oocyte or fertilized oocytereceptors and their ligands, oocyte or fertilized oocyte receptors andantibodies against them, receptors on the endometrial lining of uterusand their ligands, hormone receptors and their respective hormone,hormone receptors and antibodies directed against them, and others.Other examples may be found by referring to U.S. Pat. No. 6,265,229;U.S. Pat. No. 6,306,575 and WO 9937751, which are incorporated herein byreference. Most preferably, the adhesion molecules are antibodies,selectins, cadherins, integrins, mucin-like family, immunoglobinsuperfamily or fragments thereof. The adhesion between the selectedcells and the adhesion molecule is preferably transient, such that whenexposed to the shear rate of a flow field, preferably in the range of50-1000 s⁻¹, the cells do not bind to tightly to the adhesion molecule,but rather roll along the coated surface.

Adhesion molecules can be coated on the surface by directly physisorbing(absorbing) the molecules on the surface. Alternatively, the adhesionmolecules can be covalently attached to the surface by reacting —COOHwith —NH₂ groups on silanated glass surfaces. Another method forattachment of adhesion molecules is to first absorb or attach avidinprotein (including variants such as “Neutravidin” or “Superavidin”) tothe surface, and then reacting this avidin-coated surface with adhesionmolecules containing a biotin group. Electrostatic charge or hydrophobicinteractions can be used to attach adhesion molecules on the surface.Other methods of attaching molecules to surfaces are apparent to thoseskilled in the art, and depend on the type of surface and adhesivemolecule involved.

In a preferred embodiment, the adhesive molecule is micropatterned onthe rolling surface to improve separation, concentration, and/orpurification efficiency. The pattern is preferably a punctateddisctribution of the adhesive molecule as described by King (Fractals,12(2):235-241, 2004), which is incorporated herein by reference. Here,punctate refers to adhesion molecule concentrated in small discretespots instead of as a uniform coating, which can be in any variety ofpatterns Punctate micropatterns or other micropatterns can be producedthrough microcontact printing. This is where a microscale stamp is firstincubated upside-down with the adhesion molecule solution as a dropresting on the micropatterned (face-up) surface. Then the drop isaspirated off, the microstamp surface quickly blown dry with nitrogengas, and then the microstamp surface quickly placed face down on thesubstrate. A small 10-20 g/cm² weight can be added to the stamp tofacilitate transfer of the adhesion molecule onto the substrate. Thenthe substrate is removed and a micropattern of adhesion molecule remainson the surface.

FIG. 4 compares adhesion of flowing cells on either micropatterned oruniform adhesive surfaces. In FIG. 4A, the average rolling velocity ofcells on a micropattern is significantly lower than on a uniform surfaceof equal average density, and the micropattern is even slower than auniform surface with a much higher average density. In FIG. 4B, it isshown the rolling flux (number of adhesively rolling cells) is high onthe micropattern, is high on the uniform surface with a much higheraverage density than the micropattern, and is low on the uniform surfacewith average density matched to the micropattern. Thus, micropatterns ofadhesive molecule can be used to capture specific flowing cells muchmore effectively and efficiently than uniform adhesive surfaces. FIG. 4Cshows as picture of a punctate micropattern of adhesive molecule, 3×3micron squares of P-selectin micropatterned on tissue culturepolystyrene.

FIG. 5 shows the rolling velocity and the number of molecular adhesionbonds from a computer simulation of adhesion of a flowing cell to anadhesive surface with a (A) micropattern of molecule or (B) a uniformcoating of adhesive molecule. FIG. 5 shows that over the micropattern(“punctate”) distribution that the velocity and number of bondsfluctuates in a oscillatory, periodic way, whereas on the uniformsurface the fluctuations are random. Thus, micropatterned molecularsurfaces can be used to deliver regular, periodic surface signals toflowing cells.

FIG. 13 shows a different micropattern of adhesion molecule consistingof repeating linear stripes. Cells flowing past the micro-stripedsurface adhere to the surface and roll along. If the stripes are alignedat an angle to the direction of flow, then the cells follow the stripeand can be moved perpendicular to the flow direction. Thus, stripes ofadhesion molecules can be used to “steer” rolling cells in one directionor the other, and the cells can be led into various chambers at the endof the flow device and sorted in this way. One embodiment is to usemicrostripes of adhesion molecules to “steer” targeted adhesive cellsinto a side chamber for storage and later retrieval, while allowing mostcells or weakly adherent cells to pass through the device and not be“steered” towards the holding chamber.

In a particularly preferred embodiment, the invention exploits thenatural rolling properties of hematopoetic stem cells (HSCs), separatingthem from other blood cells in a method that is simpler, faster,cheaper, and more effective than current solutions. A novel feature isusing the differential rolling properties to separate out HSCs fromother cells in the blood. In this embodiment, the blood cells are rolledalong a surface coated with selectin proteins. The adhesion between theselectins and the HSC retards the rolling rate of HSC along the surface,while other cells rolls their normal rate. The difference in rollingrates concentrates and separates the HSCs from the other cells.

A particularly useful application of the present invention is theseparation of HSCs for use in the treatment of many cancers,hematological, and immunodeficiency diseases. The treatment of cancersand immune diseases require aggressive radiation and chemotherapy thatkills healthy bone marrow required for blood production. Bone marrow andperipheral HSC transplantation enables doctors to replace the diseasedor destroyed bone marrow with health marrow that produces normal bloodcells. The problem our device solves how to separate HSC's out of theperipheral blood supply for later readmission to the body. Our approachto the solution is to separate HSCs in flow chambers. The flow chambersurfaces are coated with selectin proteins that slow down and separateHSCs from the rest of the blood cells.

In an embodiment of the present invention, an implantable device isprovided to effect in vivo cell separation, concentration, and/orpurification in bodily fluid. The implantable device preferably containsa chamber having a surface, through which the bodily fluid passes, thatis coated with an adhesion molecule that selectively adheres to adesired cell type. The implantable device refers to any article that maybe used within the context of the methods of the invention for changingthe concentration of a cell of interest in vivo. An implantable devicemay be, inter alia, a stent, catheter, cannula, capsule, patch, wire,infusion sleeve, fiber, shunt, graft, and so on. An implantable deviceand each component part thereof may be of any bio-compatible materialcomposition, geometric form or construction as long as it is capable ofbeing used according to the methods of the invention. The literature isreplete with publications that teach materials and methods forconstructing implantable devices and methods for implanting suchdevices, including: U.S. Pat. No. 5,324,518; U.S. Pat. No. 5,976,780;U.S. Pat. No. 5,980,889; U.S. Pat. No. 6,165,225; U.S. PatentPublication 2001/0000802; U.S. Patent Publication 2001/0001817; U.S.Patent Publication 2001/0010022; U.S. Patent Publication 2001/0044655;U.S. Patent Publication 2001/0051834; U.S. Patent Publication2002/0022860; U.S. Patent Publication 2002/0032414; U.S. PatentPublication 2004/0191246; EP 0809523; EP 1174156; EP 1101457; and WO9504521, which are incorporated herein by reference.

In an embodiment, the implantable device of the present inventioncontains chamber whose surfaces are coated with adhesive molecules, suchas selectin, integrins, cadherins, mucins, immunoglobin superfamily, andcadherins, and a molecule that neutralizes the tumor-forming capacity ofthe circulating cancer cells, such as TRAIL (signal TNF-relatedapoptosis-inducing ligand), Fas ligand, and chemotherapeutic drug, (e.g.doxorubicin). The tumor neutralizing molecule is preferably connected tothe surface by a molecular stalk that can be cleaved by the cell surfacemetalloproteases and then enter the cell. In this embodiment, theimplantable device retards the rolling of cancer cells along its wall,while TRAIL kills the cancer cells slowly rolling along the coatedsurface of the device before they are released from the flow chamberback into the circulation. The device, once implanted in a patient,screens circulating blood and neutralize the tumor forming potential ofcirculating metastatic cancer cells without interruption of blood flow.This technology has the potential to provide significant benefit as anadjunct cancer therapeutic to prevent the spread of metastatic tumors,which have a significant impact on cancer related mortality anddegradation of quality of life. Furthermore, this technology has thepotential to be tuned for specific cancers to increase its effectivenessby customizing the geometric constraints, molecular interactions, andapplied therapeutic agents to optimize potency against specific cancertypes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 describes experiments using the MAD computer simulation program:(A) Dimensionless rolling velocity of a collection of nearby cells as afunction of the area fraction of adherent cells on the surface, obtainedfrom either computer simulations (solid and dashed lines) or in vitroexperiments (symbols) with sLexcoated beads rolling on P-selectin. (B)Diagram of the hexagonal array of 14 spheres used in MAD simulations ofA. (C) Measured rolling velocity of leukocytes in a live mousemicrovessel as a function of the center-to-center distance between eachcell and the nearest neighboring cell. Data is compared to a simple hrhydrodynamic scaling argument. (D) Captured image of a typicalpost-capillary venule in mouse cremaster muscle under mildlyinflammatory conditions.

FIG. 2 describes experiments using the MAD computer simulation program:(A) Representative trajectories of fluorescent tracer beads in a 40 μmvenule in mouse cremaster muscle. The arrow denotes the position of aleukocyte adherent on the vessel wall. (B) The velocity profile in themicrocirculation is approximately parabolic. (C) A random distributionof red blood cells increases the average deflection angle of the flow.The trajectory deviation angle from horizontal was found to increasemonotonically with increasing hematocrit in the numerical simulation(squares, circles), and in the in vivo experiments (stars). Note thatthe in vivo data have been reduced by a factor of 5 to account for thefact that real vessels are not mathematically smooth surfaces, and havesome inherent non-uniformity. (D) In the computational model the redblood cells were modeled as rigid spheres with volume equal to that of amature red blood cell. The case shown corresponds to 40% hematocrit.

FIG. 3 is a description of experimental methods used to study the flowof cells in vitro. (A) is a schematic diagram of a cell rolling on asurface with attached adherent molecules. (B) is a schematic of aprotocol for preparing an experimental surface.

FIG. 4 is a diagram of experimental results demonstrating the assertionof the inventors that P-Selectin can be used to selectively slow cellsas they encounter a surface coated with said protein. (A) Mean rollingvelocity v. shear stress; (B) mean rolling flux v. shear stress; and (C)a punctated pattern of adhesion molecules on a surface.

FIG. 5 is a diagram describing the interaction of cells with a coatedsurface.

FIG. 6 shows cell rolling velocity as a function of wall shear rate. (A)KG1a (blue lines) and HL60 (red lines) cells roll at similar velocitieson 0.5 μg/ml P-selectin only but in the presence of 40 μg/ml anti-CD34(dashed lines), KG1a cells roll significantly slower than HL60 cells.(B) CD34+ HSPCs (blue lines) roll significantly slower than CD34− ABMcells (red lines) on 0.5 μg/ml P-selectin±40 μg/ml anti-CD34.

FIG. 7 shows surface cell retention of CD34+ and CD34− cells. (A) KG1acells (black line) had higher retentions than HL60 cells (blue line) on0.5 μg/ml P-selectin and 40 μg/ml anti-CD34. (B) Similarly, CD34+ HSPCs(black line) had higher retentions than CD34− ABM cells (blue line) on0.5 μg/ml P-selectin only. These experiments were performed at 3 dyn/cm²for 10 minutes.

FIG. 8 shows experimental confirmation of computer simulation (A) Wepredict (green bars) and confirm with experiments (red bars) that thereshould be significant enrichment of KG1a cells on theP-selectin/antibody surface. The original concentrations (blue bars) areincluded for easier observations. (B) A more modest increase in CD34+HSPCs purity (red bars) should be possible with our current system.

FIG. 9 show determination of optimum enrichment time. While optimumenrichment should take between 10-25 minutes for KG1a cell mixtures (A),we can expect optimum enrichment to take 25-45 minutes for HSPC cellmixtures (B).

FIG. 10 is a picture depicting better separation for loading of a smallportion of the rolling surface. Loading a small portion of the surfaceinstead of the whole surface may (“bolus” system) be better forseparation.

FIG. 11 shows velocity distribution of cells at 3 dyn/cm². Experimentaldata fitted to exponentially modified Gaussian for (A) HL60/KG1a cells,(B) HSPC/CD34− ABM cells, all at 3 dyn/cm².

FIG. 12 predicts the separation abilities of a ‘bolus’ cell loadingsystem. Optimum separation should be possible within 5 minutes for allcell mixtures on a 1 mm long functional surface. Increasing the lengthof the functional surface proportionally increases the cell retentiontime and hence the tie for enrichment.

FIG. 13 shows a micropattern (punctated pattern) of adhesion moleculeconsisting of repeating linear stripes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Because perfusion flow rates and selectin density on the chamber wallcan both be used to control the average rolling velocity, computersimulations (using a computer algorithm called multiparticle adhesivedynamics (MAD) designed by the inventor specifically to study theadhesion of complex suspensions of cells to surfaces under flow) areused to determine the optimal conditions that cause CD34+ cells todownregulate their L-selectin expression while they are in close contactwith the surface.

Using the MAD computer algorithm, simulations have previously shown thatthe adhesive dynamics simulation can accurately predict the rollingvelocity and rolling fraction of cells as a function of shear rate,selectin density and species, and PSGL-1 density on the leukocyte (Kinget al., Biophys. J. 81:799-813, 2001; and King et al., Proc. Natl. Acad.Sci. USA. 98:14919-14924, 2001). Thus, the computer simulation can beused to generate design parameters that optimize the performance of theseparation device. A key parameter that the simulations will determineis the optimal delay time until the perfusion buffer is switched fromcalcium-containing to calcium-free media, in order to release the slowlyrolling CD34+ cells from the surface into the final outlet fractions(See FIG. 1).

The applicant developed this entirely new algorithm to studymultiparticle cell adhesion under flow, that builds on early work in AD.AD is a computational algorithm designed to simulate the adhesion of arigid spherical cell to a planar surface in linear shear flow (Hammer etal., Biophys. J. 62:35-57, 1992; Chang et al., Proc. Natl. Acad. Sci.USA. 97:11262-11267, 2000). The AD algorithm tracks the motion of eachmolecular bond between the cell and substrate as the cell rolls over ormoves relative to the other surface. Bonds are stochastically formed andbroken according the instantaneous probability of formation and failureas dictated by the instantaneous length (or hypothetical length in thecase of an unformed bond) of a compliant spring with endpoints on eithersurface. Other surface interactions such as electrostatic repulsion, andbody forces such as gravity, are included in the model.

To address these and other limitations of the original AD algorithm, MADwas developed. This approach is based on a boundary elements method forcalculation of the hydrodynamic mobilities of a suspension of smallparticles in a viscous fluid (Kim et al., Microhydrodynamics: Principlesand Selected Applications, Butterworth-Heinemann, Stoneham, Mass.,1991). This method, called CDL-BIEM is of general applicability, in thatit can consider any number of arbitrarily-shaped particles in a generalflow field confined by an arbitrary set of bounding surfaces. Amodification of CDL-BIEM exists to consider elastically-deformableparticles (Phan-Thien et al., ZAMP 47:672-694, 1996), and the method iscomputationally efficient insofar as being an 0(N²) process (where N isthe number of boundary elements) and is easily parallelizable (Fuenteset al., AIChE J. 38:1059-1078, 1992; and Amann et al., Eng. Anal. Bound.Elem. 11:269-276, 1993). This multiparticle hydrodynamic calculation wasfused to an improved version of AD.

Once the MAD simulation has been validated, the model was tested withobservations of leukocyte-endothelial interactions in intact venules ofan appropriate animal model of inflammation. P-selectin-mediate rollingis visualized in post-capillary venules of diameter 22-37 μm in cheekpouch of anesthesized hamsters using intravital microscopy. Rollingvelocity is found to be a strong function of the center-to-centerseparation distance to the nearest cell, and also to correlate stronglywith the number of nearby cells. These effects are beyond thatattributable to variations in vessel width or molecular expression alongthe length of the vessel. Adherent leukocytes is observed to provide anucleation site precipitating further adhesion events of free-streamcells, confirming that the hydrodynamic recruitment mechanism firstdemonstrated in simulations and cell-free experiments is indeed animportant mechanism for cell capture. These results agree with theirprevious theoretical considerations of the flow field induced bymultiple nearby cells. FIG. 2 shows representative results from the MADsimulationg, in vitro cell-free experiments and in vivo measurement ofrolling velocity, demonstrating the excellent agreement between theengineered system and the animal inflammation model.

The modification of the surface expression of CD34+ cells can beachieved by immobilizing NPPB (a broad-spectrum C1 channel inhibitor) tothe flow chamber wall. Short exposures to NPPB have been shown todecrease L-selectin levels by a factor of 2. The inventors havesuccessfully used this method to decrease the L-selectin expression onmature leukocytes, and furthermore, preliminary adhesion experimentshave confirmed that these changes in L-selectin expression significantlyaffect both average rolling velocity and rolling flux on sLeX.

In one preferred embodiment of this invention, this chemicalmodification, immobilization of NPPB on the wall of a flow chamber,alters the adhesion of this subclass of HSPC (Hematopoletic stem andprecursor cells) and alters the trafficking behavior of these cells.These results can be adapted to other surface-modifying ordifferentiation reactions.

In another preferred embodiment of the invention, the perfused cellsuspension leaves the flow chamber and is collected into the pumpsyringe and then stored after fixation until such time as the outletstream can be tested by flow cytometry to determine the extent to whichthe L-selectin expression of CD34+ cells has been successfully altered.The invention can be tested and optimized with dilute suspensions ofCD34+ cells alone, followed by test mixtures of CD34+ and whole blood.

Selectins are proteins that HSCs and white blood cells bind or stick totransiently. CD34+ stem cells are the immature stem cells and havemaximum stem cell activity, and have been shown to roll more efficiently(or slower) than CD34− stem cells, which are the more committed ordifferentiated cells. Red blood cells and platelets do not roll onselectins, while white blood cells and some tumor cells exhibit rolling.

The technology aims at exploiting the differential rolling abilities ofthese cells and accordingly designing a flow chamber coated with anoptimum distribution of selectin, molecules that can filter out thePBSCs (peripheral blood stem cells) from the remaining blood components.

United States Patent Application US20040191246, “Process For In VivoTreatment of Specific Biological Targets in Bodily Fluid,” addresses theneed for a device capable of sorting and separating useful cell typesbased on their biological properties. The patent application describesan invention comprised of “a process for the in vivo treatment of thebodily fluid of a biological organism wherein said organism is implantedwith a device, the bodily fluid is brought into contact with a bindingagent within the device and the flow velocity of at least one of thecellular components of the fluid is reduced.”

The device proposed in this application improves upon the devicedescribed in US2004/0191246 by adding the capability to manipulate adultstem cells flowing in the peripheral blood. The basic premise of thedevice is to transiently capture flowing adult HSPC from the blood, andwhile the cells are in close contact with the surface, to modify thesurface receptor presentation of the captured cell so as to modify itshoming properties. In this manner, stem cells may be redirected in thebody. Examples of improvements beyond the scope of US2004/0191246 in thepresent case include adding a recycle stream, and assembling multiplestages of flow chambers in series.

One embodiment of the device, which can be implanted in a human or ananimal, or used ex vivo, can specifically modify targeted cellsincluding cancer cells and early progenitor cells as described.

In one preferred embodiment of the current invention, cells in thecirculating blood are (i) transiently captured, (ii) chemically modifiedon their surface to alter their adhesive properties, and (iii) releasedinto the bloodstream while retaining their viability. This embodimenthas particularly preferred application in the formation of animplantable device for the selective neutralization of the tumor formingpotential of circulating metastatic cancer cells. The implantable devicepreferably contains a chamber whose surfaces are coated with an adhesivemolecule for cancer cells, preferably selectin, and a molecule thatneutralizes or kill cancer cells, preferably TRAIL, Fas ligand, orchemotherapeutic drug. Here the adhesive molecule causes the cancercells to slowly roll along the surfaces of the chamber, while the TRAIL(or other molecules that neutralizes cancer cells) neutralizes thetumor-forming capacity of the circulating cancer cells before they arereleased from the flow chamber back into the circulation. Because theTRAIL (or other molecules that neutralizes cancer cells) molecule isattached to the device surface and not freely injected into thebloodstream, it produces minimal TRAIL-related side effects andcontributes to an improved quality of life for the patient.

The device, once implanted in a patient, screens circulating blood andneutralize the tumor forming potential of circulating metastatic cancercells without interruption of blood flow. This technology has thepotential to provide significant benefit as an adjunct cancertherapeutic to prevent the spread of metastatic tumors, which have asignificant impact on cancer related mortality and degradation ofquality of life. Furthermore, this technology has the potential to betuned for specific cancers to increase its effectiveness by customizingthe geometric constraints, molecular interactions, and appliedtherapeutic agents to optimize potency against specific cancer types.

In another preferred embodiment, the device here described contains arecycle stream. Where part of the outlet stream from the device isrecycled back to the inlet stream. This effectively increases the inletconcentration of the desired cells, thus improving the concentration ofthe outlet stream.

In yet another preferred embodiment, the device here described containsa multiple stages of flow chambers in series. In this case, at least twodevices are connected in series, where the outlet stream of one devicefeeds into and inlet of the next device. Each subsequent device furtherconcentrates, separates, and/or purifies the desired cells.

One preferred embodiment of this device will consist of a glassmicrocapillary network with an inner coating of adhesive molecules inwhole or part of the network. Because the binding is not permanent, thebonds formed can dissociate quickly allowing the bound cell to “roll”when subjected to a flow stream in the microcapillary. The microcapllarysystem, also referred to as microfluidic or micro-total analysis systems(μTAS), are commonly known in the art and are disclosed in detail inU.S. Pat. No. 6,692,700 to Handique et al.; U.S. Pat. No. 6,919,046 toO'Connor et al.; U.S. Pat. No. 6,551,841 to Wilding et al.; U.S. Pat.No. 6,630,353 to Parce et al.; U.S. Pat. No. 6,620,625 to Wolk et al.;and U.S. Pat. No. 6,517,234 to Kopf-Sill et al.; which are incorporatedherein by reference. The microcapillary network is especially usefull incell separation, concentration, and/or purification of small volumesamples at high throughput.

Reproducible test data produced by the inventor shows that a precisecombination of multivalent P-selectin chimera together with anti-CD34antibodies is able to increase the difference in rolling velocitybetween HSCs and mature leukocytes from zero to a factor of two. Thisdifference in rolling velocity, with the HSCs rolling consistentlyslower over a wide range of physiological wall shear stresses (1-10dyn/cm2) will serve as the basis for a high-throughput, flow-based cellseparation process.

In one preferred embodiment of the current invention, a parallel plateflow chamber device, functionalized with a P- and E-selectin-presentingsurface to support rolling interactions of the HSPC and maturehematocyte suspensions is connected to the circulation of a patient.

Previously, the applicant has used a system to study leukocyte adhesionfocused on the cell-free assay, where leukocyte and endothelial adhesionmolecules are reconstituted in a synthetic system consisting of polymermicrospheres (model leukocytes) presenting sLe^(x), PSGL-1, or otherselectin-binding ligand (Brunk et al., Biophys. J. 72:2820-2833, 1997;and Rodgers et al., Biophys. J. 79:694-706, 2000) which serves as amodel for the construction of the current device. The lower surface of aparallel plate flow chamber is coated with P-selectin, E-selectin,L-selectin, or other adhesion molecule constitutively expressed by theendothelial cells that line blood vessels. The cell-free assay has beenshown to exhibit noisy rolling behavior similar to leukocytesinteracting with intact post-capillary venules. Cell-free experimentshave been useful in identifying the physiological role of the myriad ofreceptors and counter-receptors present on the surface of blood andendothelial cells (Goetz et al., Biophys. J. 66:2202-2209, 1994). Theapplicant has published on these experimental techniques in severalpapers (King et al., Langmuir. 17: 41394143, 2001; King et al., Biophys.J. 81:799-813, 2001; and King et al., Proc. Natl. Acad. Sci. USA.98:14919-14924,2001).

Coating of the rolling surface or chamber may be accomplished with aprotocol such as follows. The rolling surface is incubated withconcentrations of soluble P- or L-selectin (R&D Systems) ranging from2-20 μg/mL for 2 h. The coated surface will be assembled into acommercially available adhesion flow chamber (Glycotech), and connectedto a computer-controlled syringe pump (New Era Systems). Isolated HSPCwill be suspended in PBS buffer with 1 mM calcium ion and 0.5% HSA tominimize nonspecific adhesion with the surface. A mixture of cellscontaining CD34+ cells is used in the cell separation, and fed into theflow chamber with shear rates ranging from 50-1000 s⁻¹. The cells notcontaining CD34, which have been shown to exhibit weaker and moretransient adhesion to selectin-presenting surfaces, will preferentiallypass first through the flow chamber system and exit to the outletstream. Preferably, the cell mixture contains calcium because calciumion is necessary for selectin to adhere to its carbohydrate ligand. Atcertain point after flow is initiated, the inlet solution is switched tocalcium-free media which “releases” the CD34+ cells from the selectinsurface, and these cells will be mostly contained within the finalfractions of outlet suspension. The precise time at which to switchperfusion media is not yet known. However, assuming an average CD34+rolling velocity of 20 μm/s at a shear rate of 200 s⁻¹ and a usableselectin surface length of 13.5 mm, then to minimize the number of CD34+cells exiting into the calcium-containing fractions, a switchover timeof ˜14 min. should be used. This switchover time will be optimized toachieve the maximum separation of cells, by performing computersimulations of the separations experiment as described below. Therelative concentrations of CD34+ and CD34− cells can be assessed viaflow cytometry, by first treating the cell suspensions with antiCD34primary antibodies (R&D Systems, Rockville, Md.) and fluorescentsecondary antibody (Molecular Probes).

In yet another embodiment of the present invention, separation of CD34+cells from whole blood mixtures is achieved using a combination ofselectin and anti-CD34 antibody adhesion. This includes separation ofCD34+ and CD34− HSPC based on differences in selectin-mediated rolling.

In another embodiment of this invention, a variation on, and extensionof, the concept of separating cell populations that differ in CD34surface presentation but are alike in physical characteristics, mixedHSPC populations in whole blood suspensions are isolated viaselectin-mediated rolling from whole blood. In this case it will benecessary to coat the flow surface with both P-selectin (or L-selectin)and immobilized hapten-conjugated anti-CD34 monoclonal antibody (e.g.QBEND/10, IgG1, 0.5 μg/10⁶ cells). Note that the selectin molecule isnecessary since it has been demonstrated that antibody molecules aloneare insufficient to capture cells from the freestream, most likely dueto the lower rate of bond formation compared to the selecting. In thiscase common, fully differentiated leukocytes will slowly roll throughthe flow chamber due to selectin interactions, however, the moreimmature HSPC will be completely arrested due to antibody interactions.Once the mature cells are flushed from the flow chamber, the capturedHSPC must be released with a final elution step.

A preferred embodiment of this invention consists of flow chambersconstructed such that, instead of producing a well-defined parabolicvelocity profile, would better represent the complex sinusoid flow inthe bone marrow.

In one preferred embodiment, a flow chamber containing adhesionmolecules captures immature HSPC and adhesively retains them close tothe lower wall for sufficient time to chemically modify the surface ofthe cells before they are released to the bulk flow at the downstreamedge of the functional flow chamber.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following example isgiven to illustrate the present invention. It should be understood thatthe invention is not to be limited to the specific conditions or detailsdescribed in this example.

EXAMPLE 1

In order to establish protocol without sacrificing precious HSPCs, weutilized a model system where CD34⁺ KG1a cells represented the HSPCs andCD34⁻ HL60 cells represented the CD34⁻ ABM cells. The KG1a/HL60 modelwas used to determine an optimum P-selectin concentration for subsequentHSPC experiments. We initially found that KG1a and HL60 cells rolled atvery similar velocities at all P-selectin concentrations tested so,based on the data from Eniola et al. (2003), we co-immobilized anti-CD34antibody together with the P-selectin and found that, at 0.5 μg/mlP-selectin and 40 μg/ml anti-CD34, there was a significant differencebetween the rolling velocities of the two cells (FIG. 6A). This moreclosely represented previous findings that HSPCs tend to roll slowerthan CD34⁻ cells on selecting, which was further confirmed by our ownHSPC/CD34⁻ ABM cells experiments using 0.5 μg/ml P-selectin (FIG. 6B).The presence of the antibody had little effect on the rolling velocityof the ABM cells so it was not used in subsequent experiments using ABMcells.

EXAMPLE 2

Cell retention as a function of time was also determined for both cellmodels at a shear stress of 3 dyn/cm² for 10 minutes. Cells wereinitially loaded over the entire surface and allowed to settle for 40 sfor KG1a/HL60 cells, and 2 minutes for ABM cells, based on the Stokessettling velocity of the cells of interest. We found that KG1a Cells hada higher accumulation than HL60 cells on the P-selectin/antibody surfaceand similarly, there was higher retention of HSPCs than Cd34⁻ ABM cellson the P-selectin surface (FIG. 7).

We were able to use this data to predict and confirm with experimentsthat there would be significant enrichment of KG1a cells for KG1a/HL60cell mixtures ranging from 10-50% KG1a cells. Predictions usingphysiologic ABM concentrations of 1-5% HSPC showed more modestimprovements and were not confirmed experimentally (FIG. 8).

We extended the prediction to determine the length of time for optimumenrichment, i.e., the time for purity and retention to be equal. Wedetermined that while optimum enrichment would take less than 25 minuteswith KG1a/HL60 cell mixtures, it would take over 30 minutes for modestenrichment of HSPC (FIG. 9).

EXAMPLE 3

As mentioned before, we established conditions for determining theeffectiveness of our system based on recommendations from Johnsen et al(1999)—Cell purity >80-90%, Cell retention >50% and optimum separationwithin 30 minutes. It was evident that our current system neededsignificant improvements to achieve these preliminary goals, so weinvestigated whether our cell loading system was optimized for this typeof separation. Instead of loading the entire surface, only a smallportion (<10%) of the surface would be used for the initial cell loadingstep so that the device could make use of the natural tendency of thecells to separate based on rolling velocity (FIG. 10).

We used an exponentially modified Gaussian (EMG) distribution todescribe the velocity distribution of cells at 3 dyn/cm² (FIG. 11). Thepeak to peak resolution for HL60/KG1a cells and HSPC/CD34⁻ ABM cells wasabout 0.4, corresponding to about 40% cross contamination. Coupled withthe cell retention data obtained at t=0 s, we were able to predict theoptimum cell enrichment possible with 10-50% KG1a cell mixtures and 1-5%HSPC cell mixtures, assuming a functional length of 1 mm (FIG. 12). Inboth cases, optimum cell separation should be possible within 5 minuteswith significant improvements in purity over our current loading system.

Since we envision the final device as a multistage device, we expecteven higher purities and cell recovery >50% should be likely sincedetached CD34⁺ cells can be recaptured in subsequent stages. Ourpreliminary experiments and prediction confirm that cells can beseparated based on differential rolling velocities, and while we arelimited by the current design of our experimental system, proper designand manufacturing techniques could make this device a reality. Wecontinue to investigate new ways of improving the theoreticaleffectiveness of the system and search for alternative experimentalmethods for testing our separation predictions.

Although certain presently preferred embodiments of the invention havebeen specifically described herein, it will be apparent to those skilledin the art to which the invention pertains that variations andmodifications of the various embodiments shown and described herein maybe made without departing from the spirit and scope of the invention.Accordingly, it is intended that the invention be limited only to theextent required by the appended claims and the applicable rules of law.

1. A method for separating, concentrating, and/or purifying a particular cell type from a mixture of cells comprising the steps of providing a flow surface containing an adhesive molecule that selectively binds with the particular cell type; and flowing the mixture of cells on the flow surface.
 2. The method of claim 1, wherein the particular type of cell is selected from the group consisting of CD34+ cell and hematopoietic stem and precursor cell (HSPC).
 3. The method of claim 1, wherein the substance is selected from the group consisting of NPPB, P-selectin, L-selectin, E-selectin, antibody specific against the particular cell type, cadherins, integrins, mucin-like family, immunoglobin superfamily and fragments thereof.
 4. The method of claim 1, wherein the flow surface is part of a separation chamber.
 5. The method of claim 1, wherein the flow surface is part of a microcapillary network.
 6. The method of claim 1, wherein the wall shear stress is about 1-10 dyn/cm².
 7. The method of claim 1, wherein the particular cell type has a rolling speed less than half the rolling speed of the other cells in the mixture of cells.
 8. An implantable shunt for directing stem cell trafficking comprising a flow chamber, wherein a wall of the chamber contains an adhesive molecule that selectively binds with the stem cell.
 9. The implantable shunt of claim 8, wherein the stem cell is CD34+ hematopoetic stem cell.
 10. The implantable shunt of claim 8, wherein the molecule is selected from the group consisting of NPPB, P-selectin, L-selectin, E-selectin, antibody specific against the particular cell type, cadherins, integrins, mucin-like family, immunoglobin superfamily and fragments thereof.
 11. An implantable device for neutralizing tumor cells comprising a flow chamber, wherein a wall of the chamber contains an adhesive molecule that selectively binds with the tumor cells and a molecule that neutralizes the cancer cells.
 12. The implantable device of claim 11, wherein the adhesive molecule is selectin.
 13. The implantable device of claim 11, wherein the molecule that neutralizes the cancer cells is selected from the group consisting of TRAIL, Fas ligand, and chemotherapeutic drug.
 14. The implantable device of claim 11, wherein the molecule that neutralizes the cancer cells induces apoptosis in the tumor cells.
 15. A method for neutralizing tumor cells comprising the steps of providing a flow surface containing an adhesive molecule that selectively binds with the tumor cells and a molecule that neutralizes the cancer cells; and flowing the tumor cells on the flow surface.
 16. The method of claim 15, wherein the adhesive molecule is selected from the group consisting of NPPB, P-selectin, L-selectin, E-selectin, antibody specific against the particular cell type, cadherins, integrins, mucin-like family, immunoglobin superfamily and fragments thereof.
 17. The method of claim 15, wherein the molecule that neutralizes the cancer cells is selected from the group consisting of TRAIL, Fas ligand, and chemotherapeutic drug.
 18. The method of claim 15, wherein the molecule that neutralizes the cancer cells induces apoptosis in the tumor cells.
 19. The method of claim 15, wherein the flow surface is part of a separation chamber.
 20. The method of claim 15, wherein the flow surface is implanted in the circulatory system of a patient. 